JPH048036B1 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention is a method for producing L(+)-β-hydroxy fatty acids, which have two different functional groups and are useful synthetic intermediates for pharmaceuticals, agricultural chemicals, etc., using microorganisms. More specifically, L(+)-β-hydroxybutyric acid, which is derived from a microorganism belonging to the Candeida genus or Debaryomyces genus and having the ability to convert butyric acid to L(+)-β-hydroxybutyric acid, is used as a single carbon source. A mutant strain that does not grow or grows poorly on a nutrient medium containing saturated fatty acids with 4 or 5 carbon atoms,
Alternatively, L having 4 or 5 carbon atoms is produced by acting on α,β-unsaturated fatty acids or alcohols.
The present invention relates to a method for producing L(+)-β-hydroxy fatty acids, which comprises collecting (+)-β-hydroxy fatty acids. (Prior art and problems) Regarding the production method of L(+)-β-hydroxybutyric acid having 4 carbon atoms, ethyl acetoacetate is asymmetrically reduced using baker's yeast to produce ethyl L(+)-β-hydroxybutyrate. Although a method for obtaining it is known (Mori et al.; Journal of the Chemical Society of Japan, No. 9, p. 1315, 1983), it requires a large amount of yeast and is difficult to imagine as an economical method.
On the other hand, regarding the production method of L(+)-β-hydroxyvaleric acid having 5 carbon atoms, a method is known in which ethyl β-ketovalerate is obtained as D(−)-ethyl β-hydroxyvalerate by reduction with baker's yeast. [Freter et al., Helvetica Chimica Acta (G,
Frater, Helvetica Chimica Acta) 62 volumes, 2829
Page, 1973], the configuration is reversed. The present inventors previously discovered that L(+)-β-hydroxyvaleric acid could be produced from valeric acid using Candida rugosa IFO 1542 (Japanese Patent Publication No. 59-53838), but These microorganisms metabolize linear L(+)-β-hydroxy fatty acids very easily, resulting in extremely low yields and are not suitable for mass production. Therefore, the present inventors have developed an inexpensive and efficient L
As a result of research to develop a method for producing (+)-β-hydroxy fatty acids, it was found that butyric acid was produced by L.
By mutating and improving microorganisms that have the ability to convert L(+)-β-hydroxybutyrate to L(+)-β-hydroxybutyric acid and inducing a mutant strain that does not grow in a nutrient medium with L(+)-β-hydroxybutyrate as the sole carbon source, butyric acid , L(+)-β-hydroxybutyric acid is produced in high yield from crotonic acid or n-butyl alcohol, and L(+)-β-hydroxyvaleric acid is produced from valeric acid, 2-pentenoic acid or n-amyl alcohol. We have already filed a patent application (Japanese Patent Application No. 59-44753), but after further investigation, we found that a mutant strain of a microorganism belonging to the genus Candeida or the genus Debaryomyces has been found to have this ability. discovered the strain and completed the present invention. (Means for Solving the Problems) In carrying out the present invention, Candida pararugosa IFO 0966, Debaryomyces hansenii, and Debaryomyces hansenii are used as parent strains for obtaining mutant strains.
hansenii) IFO 0032 etc., but butyric acid is L(+)
-β-Hydroxybutyric acid and valeric acid L(+)-β-
Any microorganism that has the ability to convert hydroxyvaleric acid can be used. As a method for converting microorganisms into L(+)-β-hydroxy fatty acids by interacting with a substrate, microorganisms are cultured in a nutrient medium, and microorganisms are separated from the culture solution or culture solution and the microorganisms are suspended. There are methods such as preparing a suspension and reacting it with a substrate, or culturing microorganisms in a medium to which a substrate is added, and then reacting the microorganisms with the substrate. The isolated bacterial cells can also be used in a state where they are immobilized with a water-insoluble polymer or the like. By supplying an energy source that can be used by microorganisms during a contact reaction between microorganisms and a substrate, L(+)
-The productivity of β-hydroxy fatty acids is improved. Preferred energy sources in this case include glucose, glycerol, and the like. Since ordinary microorganisms have a fast metabolic rate of L(+)-β-hydroxy fatty acids, the amount of L(+)-β-hydroxy fatty acids accumulated is extremely small, making it difficult to produce them economically. Therefore, in order to efficiently accumulate large amounts of L(+)-β-hydroxy fatty acids, it is necessary to use mutant strains that have a slow metabolic rate or do not metabolize L(+)-β-hydroxy fatty acids, in other words, use L(+)-β-hydroxy fatty acids as a single carbon source. It is advantageous to use mutant strains that do not grow or grow poorly on nutrient media. Artificial mutation or natural mutation is used to obtain such mutant strains, but artificial mutation is usually used for efficient mutation. As an artificial mutation method,
Radiation, ultraviolet irradiation, gamma ray treatment, and treatment with mutagenic agents such as N-methyl-N-nitro-N'-nitrosoguanidine (NTG) are used. As a specific example, the present inventors have demonstrated that L(+)-β-
An example of a mutation method using NTG used to obtain a mutant strain that does not metabolize hydroxy fatty acids is as follows. However, the method is not limited to this method as long as the desired mutant strain can be obtained. Preservation slant (Quiandida Pargosa)
1 platinum loop from IFO 0966) 40g of glucose,
( _NH4 ) _2HPO4 13g, _KH2PO47g , _{MgSO4 ・
7H2O0.8g} , _ZnSO4・_7H2O60mg , _FeSO4・
_7H2O90mg , _CuSO4・_5H2O5mg , _MnSO4・
S medium consisting of 10 mg of 4H ₂ O, 0.1 g of NaCl, 1 mg of biotin, 2 mg of thiamine-HCl, 1 portion of water, and PH7.2.
Pour 30ml into a 500ml flask, inoculate, and inoculate at 30℃ for 20 minutes.
Cultured with shaking for hours. After washing 1.5 ml of the culture solution with 0.5 M phosphate buffer (PH 7.0), it was suspended in 3 ml of 0.5 mg/ml NTG solution and left at 4° C. for 60 minutes. Afterwards, the solid plate medium C was washed with the same buffer solution (20 g of glucose, 5 g of yeast extract, 10 g of meat extract, 10 g of beptone, agar) and was washed with the same buffer three times.
(20 g, 1 part water, PH7.0) and colonies were allowed to appear. This colony was replicated onto B medium of PH 7.0 containing 10 g of butyric acid and 20 g of agar instead of glucose in S medium, and cultured at 30° C. for 2 days. Bacteria that grew poorly on this B medium (strains that could not assimilate butyrate) were selected.
The non-butyric acid-assimilating strain thus obtained was planted on S medium, cultured at 30°C for 48 hours, and the production of L(+)-β-hydroxybutyric acid was determined by gas chromatography [Hasegawa et al.; Journal of Fermentation Technology (J.Ferm.Tech.) Vol. 59, 257
Page, 1981], and L(+)-β-hydroxybutyric acid producing strains were selected. The mutant strains selected in this way are grown on a nutrient medium containing L(+)-β-hydroxybutyric acid as the sole carbon source (for example, a medium in which L(+)-β-hydroxybutyric acid is added instead of glucose in S medium). It can be used in the present invention because the growth in this species is significantly reduced. Mutations of microorganisms of other genera can be carried out using similar techniques, and examples of mutant strains obtained by the above method for carrying out the present invention include Candida pararugosa KT 84015,
Debaryomyces hansenii
hansenii) KT 84016 etc. There was almost no difference in the properties of this mutant strain from the parent strain, but as shown in Table 1, a significant difference was observed in the ability to assimilate L(+)-β-hydroxy fatty acids.

[Table] +++++: Good growth, -: Poor growth These mutant strains have been deposited with the following numbers at the Institute of Microbiology, Agency of Industrial Science and Technology, as shown in Table-2.

【table】

[Table] The microorganism culture medium used in the present invention includes carbon sources such as glucose and glycerol, nitrogen sources such as inorganic and organic nitrogen-containing compounds such as ammonia, ammonium sulfate, peptone, and casamino acids, potassium phosphate, magnesium sulfate, etc. Inorganic salts necessary for growth, vitamins such as biotin, and various other nutritional sources used in ordinary microbial culture can be appropriately mixed and used as necessary. For culturing, bacteria are inoculated into a sterilized medium and incubated at a temperature of 20 to 45°C and maintained at a pH of 6 to 9.
Carry out aerobic culture such as aeration and shaking for ~10 days. There is bacterial cell production in the early stage of culture, and then L(+)-
Production of β-hydroxy fatty acids takes place. In addition, by supplementing glucose or glycerol as an energy source during the production of L(+)-β-hydroxy fatty acids, L(+)-β-
Production of hydroxy fatty acids takes place. The substrate may be added to the medium from the early stage of culture or after the bacterial cells have grown. L(+) produced from culture solution or bacterial cell reaction solution
-β-hydroxy fatty acids can be recovered by means commonly used for recovering β-hydroxy fatty acids. For example, after removing bacterial cells, L
After concentrating the (+)-β-hydroxy fatty acid-containing liquid, the pH is lowered to below 2.5 with sulfuric acid, etc., and then extracted with ether, ethyl acetate, etc. After removing the solvent, L(+)-β -Hydroxy fatty acids can be obtained. Since L(+)-β-hydroxy fatty acids are not very stable substances, the L(+)-β-hydroxy fatty acids obtained by the above solvent extraction etc. are dissolved in alcohol such as methanol or ethanol, and in the presence of a catalyst such as sulfuric acid. It can be easily converted to an alkyl ester by heating at a low temperature. If this product is distilled, a highly purified L(+)-β-hydroxy fatty acid ester can be obtained in a high yield, and this ester is relatively stable. (Effects of the Invention) According to the present invention, optically active L(+)-β-hydroxybutyric acid and L(+)-β-hydroxyvaleric acid can be industrially advantageously produced, and useful synthetic intermediates for pharmaceuticals and the like can be supplied. It became like that. (Examples) The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these Examples. Example 1 Glucose 40g, yeast extract 5g,
(NH ₄ ) ₂ HPO ₄ 13g, KH ₂ PO ₄ 7g, MgSO ₄・
_7H2O0.8g , _ZnSO4・_7H2O60mg , _FeSO4・
_7H2O90mg , _CuSO4・_5H2O5mg , _MnSO4・
4H ₂ O 10mg, NaCl 0.1g, butyric acid 10ml or valeric acid 10
ml (per unit) of the medium with NaOH.
PH7.2, put this 3 in a 5-volume mini-jar fermenter, sterilize it, and then incubate Candida pararugosa IFO 0966 or its mutant strain KT 84015, or Debaryomyces hansenii IFO.
Plant 0032 or its mutant KT 84016 and aerate.
The cells were cultured at 1vvm, stirring at 500 rpm, and 30°C for 4 days. During the culture, the pH was maintained at 7.0, and 60 g of glucose was added every day. After the completion of the culture, the produced L(+)-β-
As a result of measuring hydroxybutyric acid or L(+)-β-hydroxyvaleric acid, accumulation as shown in Table 3 was observed.

【table】

[Table] In addition, when no substrate is added, L(+)-β-hydroxybutyric acid or L(+)-β
-No accumulation of hydroxyvaleric acid was observed. After sterilizing the culture solution obtained under the above culture conditions, the supernatant solution was concentrated to 50 ml under reduced pressure, and then the pH was adjusted to 2.0 with sulfuric acid.
This was extracted three times with 700 ml of ethyl acetate, and after removing the solvent, each L(+)-β-hydroxy fatty acid was obtained in the form of a yellow oil. This was purified by vacuum distillation and Table 4
A colorless oily L(+)-β-hydroxy fatty acid was obtained. By NMR, gas chromatography, and specific rotation measurement after conversion to methyl ester, butyric acid yields L(+)-β-hydroxybutyric acid, and valeric acid yields L(+)-β-hydroxyvaleric acid. This was confirmed.

[Table] Example 2 Medium 3 from which fatty acids had been removed from the medium shown in Example 1 was placed in a 5-volume mini-jar fermenter, and after sterilization, Candeida parargosa KT 84015 or Debaryomyces hansenii KT 84016 was used.
was inoculated and cultured at 30°C for 24 hours with aeration of 1vvm and stirring at 500rpm. To each of the culture solutions, 15 g of crotonic acid, n-butyl alcohol, 2-pentenoic acid, or n-amyl alcohol was added, and 60 g of glucose was added every day while maintaining the pH at 7.0, and the reaction was continued for an additional 3 days. After the reaction was completed, the produced L(+)-β-hydroxy fatty acids were measured by gas chromatography, and as a result, accumulation of each L(+)-β-hydroxy fatty acid as shown in Table 5 was observed.

[Table] Example 3 Similar to Example 1, Candeida Paralgoza
After culturing KT 84015 or Debaryomyces hansenii KT 84016, 24, 48,
60g glucose or glycerol at 72 hours
60 g each was added and cultured for 96 hours while maintaining the pH at 7.0. The amount of L(+)-β-hydroxy fatty acid produced in the culture solution after completion of the culture was as shown in Table 6.

【table】

Claims (1)

[Scope of Claims] 1. Belongs to the genus Candida or Debaryomyces, and contains butyric acid in L
A mutant strain derived from a microorganism that has the ability to convert L(+)-β-hydroxybutyric acid to L(+)-β-hydroxybutyric acid that does not grow or grows weakly in a nutrient medium containing L(+)-β-hydroxybutyric acid as the sole carbon source. L (+ )-Method for producing β-hydroxy fatty acid. 2. The microorganism is Candida pararugosa or Debaryomyces hansenii.
Claim 1, which is a mutant strain derived from
Manufacturing method described in section. 3 The saturated fatty acid used as a substrate is butyric acid or valeric acid, the α,β-unsaturated fatty acid is crotonic acid or 2-pentenoic acid, and the alcohol is n-butyl alcohol or n-amyl alcohol,
3. The manufacturing method according to claim 1 or 2, wherein the product to be manufactured is the corresponding L(+)-β-hydroxybutyric acid or L(+)-β-hydroxyvaleric acid. 4. The manufacturing method according to claim 1, 2, or 3, which comprises culturing microorganisms in a nutrient medium and applying a substrate to the obtained culture solution. 5. The manufacturing method according to claim 1, 2, or 3, which comprises culturing in a medium to which a substrate is added, and allowing the substrate and microorganism to interact. 6. Cultivate microorganisms in a nutrient medium, isolate microorganism cells from the resulting culture solution, and prepare a cell suspension;
Claim 1, in which a substrate acts on it;
The manufacturing method according to item 2 or 3. 7. The production method according to any one of claims 1 to 6, wherein an energy source that can be used by microorganisms is supplied when the microorganisms interact with the substrate. 8. Claim 7, wherein the energy source that can be used by microorganisms is glucose or glycerol.
Manufacturing method described in section.